Process for preparing purified nucleic acid and the use thereof

ABSTRACT

The invention relates to a nucleic acid preparation with a content of below 1% protein, preferably below 0.1% protein, free of ethidium bromide, phenol, cesium chloride and detergents based on octyl phenol poly(ethylene glycol ether) n  and with a content of below 1EU/mg DNA of endotoxins. Said preparation is suitable as a drug particularly in gene therapy.

This application is a divisional of Ser. No. 11/389,167 filed Mar. 27,2006, which is a continuation of U.S. Ser. No. 10/771,371 filed Feb. 5,2004, which is a divisional of U.S. Ser. No. 09/117,537 filed Aug. 4,1998, now U.S. Pat. No. 6,750,333, which is a 35 USC 371 ofPCT/EP97/00321 filed Jan. 24, 1997.

The invention concerns the preparation of purified nucleic acid and itsuse especially in gene therapy.

Replicatable nucleic acid is usually produced by amplifying replicatableplasmid DNA in gram-negative bacteria such as e.g. E. coli. After lysisof the biomass (usually alkaline lysis with lysozyme or ultrasound), itis centrifuged and the supernatant is shaken out with phenol.Subsequently an ultracentrifugation on a caesium chloride gradient iscarried out (Birnboim & Doly, Nucleic Acid Res. 7 (1979) 1513-1523,Garger et al., Biochem. Biophys. Res. Comm. 117 (1983) 835-842).However, such preparations contain endotoxins, phenol, caesium chlorideand/or ethidium bromide as a dye.

A further process is described in the QIAGEN® Plasmid Handbook (QiagenInc., Chatsworth, USA) and EP-B 0 268 946. According to this process thecell lysate obtained after a conventional lysis is chromatographed onQIAGEN®-TIP, which contains QIAGEN® resin (a support material based onsilica). The disadvantage of this process is that DNA binding proteinsare not completely detached from the DNA and therefore the purifiedplasmid fraction contains proteins and in particular endotoxins (fromthe membrane of the gram-negative host cells) in considerable amounts.

In another process after alkaline lysis of the E. coli biomass thecentrifugation supernatant is chromatographed according to Birnboim &Doly under high salt conditions over anion exchange columns (e.g.Mono-Q, Source-Q from Pharmacia, Macroprep-Q from BioRad, Poros-Q fromPerseptive Biosystems or HyperD-Q from Biosepra, cf. Chandra et. al.,Analyt. Biochem. 203 (1992) 169-172; Dion et al., J. Chrom. 535 (1990)127-147). Also in this case the purified plasmid fraction containsproteins and in particular endotoxins in considerable amounts.

In another process after alkaline lysis and subsequent phenol/chloroformextraction it is possible to chromatograph by gel filtration (McClung &Gonzales, Analyt. Biochem. 177 (1989) 378-382; Raymond et al., Analyt.Biochem. 173 (1988) 125-133). Even after this purification the plasmidpreparation contains impurities and in particular phenol.

A process for the isolation and purification of nucleic acids for use ingene therapy is described in WO 95/21177 in which the purification isessentially carried out by centrifugation, filtration, affinitychromatography or chromatography on an inorganic chromatographicmaterial with subsequent chromatography on an ion exchanger. Anadditional removal of endotoxins can then be achieved according to WO95/21177 when the nucleic acid is treated with an endotoxin removalbuffer which contains 10% Triton®X100 and 40 mmol/l MOPS buffer(3-morpholino-1-propanesulfonate buffer). A disadvantage of this processis that the nucleic acid purified in this manner contains impurities ofTriton® and MOPS buffer. Although endotoxins can be removed by thisprocess to a content of ca. 100 EU/mg DNA (Qiagen News 1/96, 3-5), it isnot possible to remove endotoxins to a greater extent by this process.

However, for a therapeutic application such as for example for genetherapy a nucleic acid preparation is required which is as free aspossible of all impurities (in particular substantially free ofendotoxins). Above all the endotoxin content of plasmid preparations hasbeen hitherto an unsolved problem as described for example by Cotten etal., Gene Therapy 1 (1994) 239-246. A reduced endotoxin content (ca. 100EU/mg DNA) can only be achieved by the state of the art such as forexample according to WO 95/21177 if the nucleic acids are treated withnon-ionic detergents such as e.g. Triton (endotoxin removal buffer fromWO 95/21177). However, Triton® has a biological action such as e.g. lungchanges or reduction of blood pressure (Fiedler, “Lexikon der Hilfstoffefür Pharmazie und Kosmetik und angrenzende Gebiete (Band 9, 3rd edition,1989, Editio Cantor, DE)). The MOPS buffer which is additionallyrequired also contains a substance that is problematic with regard to atherapeutic application.

The invention provides a nucleic acid preparation, preferably a plasmidDNA, of high purity in which endotoxins are substantially removed andpreferably without ethidium bromide, phenol, caesium chloride, polymyxinor non-ionic detergents and also provides a simple and effective processfor purifying such nucleic acids in particular for removing endotoxins.

The invention concerns a nucleic acid that can be replicated ingram-negative bacteria, preferably a plasmid DNA with a content of lessthan 1% protein, preferably less than 0.1% protein and a content of lessthan 1 EU/mg DNA, preferably 0.01-0.1 EU/mg DNA of endotoxins. Thisplasmid DNA is preferably free of ethidium bromide, phenol and caesiumchloride, free of detergents based on octylphenolpoly(ethylene glycolether)_(n) such as Triton® detergents and also free of MOPS buffersubstance and RNAse.

Amplification is understood as an increase in the copy number of anucleic acid (in particular DNA and plasmid DNA) based on thereplication of a vector. In this process numerous copies are producedfrom a template. A vector is replicated which represents the nucleicacid or which contains the cloned nucleic acid.

A plasmid DNA is understood as an extrachromosomal DNA duplex molecule.The size of a DNA plasmid is usually 1 to more than 200 kb and one toseveral hundred copies are present in host cells. Plasmid DNA is usuallyamplified in gram-negative bacteria such as e.g. E. coli andsubsequently isolated. Plasmids are often used to construct cloningvectors, for the transfection of prokaryotic and eukaryotic cells. Atherapeutic use is of especial importance in connection with in vivo andex vivo gene therapy. Plasmid DNA that is used therapeuticallypreferably has a length of 5 to 20 kb, particularly preferably 5-10 kband is double-stranded. The plasmid DNA can be linearized or circularlyclosed. Preferably DNA is used that is essentially circularly closed.

Consequently the invention additionally concerns a pharmaceuticalcomposition containing a nucleic acid according to the invention,preferably plasmid DNA, in a therapeutically effective amount andoptionally additional pharmaceutical auxiliary substances, fillers oradditives.

Endotoxins are lipopolysaccharides from gram-negative bacteria.Endotoxins can have a pyrogenic effect in mammals and induce anendotoxin shock. The main toxic component of endotoxins is lipid A, thepolysaccharide moiety mediating the water solubility and the lipidmoiety having the toxic effect. The biological effect of endotoxins inmammals are in particular a hypersensitization as well as otherreactions which are accompanied by fever.

Plasmid DNA is amplified by standard methods in E. coli i.e. agram-negative bacterium. After fermentation the biomass obtained in thisprocess is lysed and the cells are lysed. In this process the endotoxinsare released from the cell membrane. This means that after amplificationof nucleic acids, in particular of plasmid DNA, in gram-negativebacteria and in particular in E. coli it is necessary to removeendotoxins if it is intended to use this plasmid DNA therapeutically.

Depending on the application doses of 50 μg to 10 mg and more are usedor planned for a therapeutic application of replicatable nucleic acids,in particular of plasmid DNA. The dose amount depends on the disease andtype of administration. In an aerosol, e.g. for the treatment of cysticfibrosis, doses of 400 μg and more are used. This applies likewise toplasmid DNA encapsulated in a lipid complex (e.g. in liposomes). Inorder to provide such amounts of replicatable nucleic acid that can beused therapeutically, it is necessary to produce the replicatablenucleic acid on a large scale. For this fermentation preparations areexpedient with 1-5 kg biomass from which 1-5 g nucleic acid can beisolated.

The invention also concerns a process for the production of a plasmidDNA with a content of less than 1 EU/mg DNA, preferably 0.01-0.1 EU/mgDNA of endotoxins which is characterized in that plasmid DNA isreplicated in gram-negative bacteria, the biomass is lysed and thesoluble components are chromatographed on hydroxylapatite andsubsequently the said plasmid DNA is isolated. Before chromatography onhydroxylapatite it is preferably to carry out an ion exchangechromatography which essentially removes RNA and foreign proteins. Thiscan optionally remove further impurities and achieve a content ofnucleic acid of less than 1% protein, preferably less than 0.1% protein,free of ethidium bromide, phenol and caesium chloride. Such apreparation is also preferably free of detergents based on octylphenolpoly(ethylene glycol ether)_(n) and MOPS buffer substance.

The process according to the invention enables numerous impurities to beavoided or removed which plasmid DNA contains if it is produced by aprocess familiar to a person skilled in the art. Above all it issurprisingly possible to drastically reduce the endotoxin content in asimple manner.

In the process according to the invention an outstanding removal ofendotoxins is achieved by the chromatography with hydroxylapatite. Thisis all the more surprising since chromatography on hydroxylapatite isonly used in the literature to separate DNA and RNA (Johnson & Ilan,Analyt. Biochem. 132 (1983) 20-25).

The chromatographic effect of hydroxylapatite is essentially based onthe interaction between calcium²⁺ groups and the negative charge of thenucleic acid to be purified and to a lesser extent on the interaction ofthe nucleic acid to be purified with PO₄ ³⁻ groups on the surface ofcrystalline hydroxylapatite (cf e.g. Protein Purification Methods, Ed.by Elv. Harries and S. Angal, Oxford University Press 1989, 238-244).Chromatography on hydroxylapatite can be essentially referred to as anion exchanger step for nucleic acids in which the bound DNA cannot beeluted from the hydroxylapatite matrix by a simple increase of ionicstrength (e.g. NaCl) but rather by increasing the concentration,preferably of phosphate or citrate, divalent metal ions and/or EDTA.

In the process according to the invention endotoxins and the nucleicacid to be purified are firstly bound to hydroxylapatite primarily viadipole-dipole interactions in the chromatography on hydroxylapatite(e.g. HA-ceramic, BioGel HPHT, Bio-Gel HT/HTP from Biorad Del.,HA-Ultrogel from IBF or HA spheroidal from BDH, Macrosorb C fromSterling Organics). The equilibration is usually carried out at neutralpH in phosphate buffer. A denaturing agent is preferably added, as inthe subsequent washing of the column. Surprisingly it is possible todisplace the nucleic acid from its binding to hydroxylapatite withphosphate, citrate or calcium ions whereas the endotoxins remain bound.Instead of calcium, the displacement of the nucleic acid from itsbinding to hydroxylapatite can be achieved with other divalent metalions which can replace calcium in the apatite such as e.g. Mg, Fe, Mn.For the elution the ion concentration is preferably 100 mol/l or more.Ion concentrations between 100 and 500 mmol/l or 200 and 500 mmol/l areparticularly preferred. A solution containing phosphate ions (e.g.phosphate buffer) is particularly preferably used. Before the elution(without denaturing agent) it is expedient to wash (with denaturingagent). It has turned out to be advantageous to for example use aphosphate of sulfate solution (100-200 mmol/l) to which a denaturingagent (e.g. urea or guanidine hydrochloride) has been added at aDNA-denaturing concentration (e.g. 6 mol/l urea).

In a preferred embodiment an ultrafiltration is additionally carried outafter the chromatography on hydroxylapatite.

In order to produce the nucleic acid according to the invention theplasmids which represent or contain the nucleic acid are usuallyamplified in gram-negative bacterial cultures. Such methods are known toa person skilled in the art and are described for example by Sambrook etal. (1989), Molecular Cloning: A Laboratory Manual, 2nd edition, ColdSpring Harbor Laboratory Press and by F. M. Ausubel et al., eds. (1991),Current Protocols in Molecular Biology, Wiley Interscience, New York.For this the bacterial cultures which contain the plasmids are firstlysubcultured and subsequently cultured in a suitable medium optionallywith addition of a selection agent.

The biomass is also lysed by methods familiar to a person skilled in theart (mechanical or enzymatic lysis, see e.g. Birnbolm and Doly, NucleicAcids Research 7 (1979) 1513-1423) without addition of RNAse. It ispossible to omit shaking out with phenol if the proteins are separatedby chromatography on an anion exchanger. After lysis and separation ofthe insoluble components, preferably by centrifugation and filtrationover a filter candle (5 μm pores), the cell supernatant is preferablyfirstly chromatographed on an anion exchanger to remove proteins.Suitable anion exchangers are anion exchangers based on agarose such asfor example Q-Sepharose. Other suitable anion exchangers are based onpolymethacrylate (Macroprep/Bio-Rad, Germany),polystyrene/divinylbenzene (Poros/Perseptive, HyperD/Biosepra,Source/Pharmacia) or silica gel on the surface of whichdiethylaminoethyl (DEAE) or dimethylaminoethyl (DMAE) groups are forexample bound.

In order to optimize the purification effect, the nucleic acid is elutedby means of a high salt gradient e.g. NaCl gradient (preferably 0.65mol/l-0.85 mol/l) in TE buffer. This surprisingly enables numerousimpurities (RNA, protein) to be separated in one step.

It is also preferable to carry out an additional isopropanol/ethanolprecipitation, preferably after the hydroxylapatite chromatography, tominimize the bioburden and for desalting. Subsequently the nucleic acidaccording to the invention can be bottled under sterile. conditions.

The following examples describe the invention in more detail.

EXAMPLE 1 Isolation of Nucleic Acid from E. coli Biomass

E. coli biomass containing plasmid DNA is lysed by an alkaline lysis andthe released plasmid DNA is chromatographed over Q-Sepharose andHA-Ceramic. The eluate is desalted by an isopropanol/ethanolprecipitation and concentrated and the plasmid DNA precipitate isresuspended in TE buffer.

Resuspension buffer: 50 mmol/l Tris/HCl, 10 mmol/EDTA-Na₂, pH 8.0±0.2

Potassium acetate buffer: 3 mol/l potassium acetate buffer pH 5.5

60 g biomass (wet, E. coli) from the fermenter is filled intodepyrogenized centrifuge beakers. 750 ml resuspension buffer is addedand it is stirred slowly (ca. 35 rpm) for at least 24 hours at 5±4° C.until the biomass is completely suspended. During this process thetemperature, of the suspension is slowly increased to 25° C.

750 ml 0.2 mol/l NaOH/1% SDS is added to the suspension while stirringat ca 80 rpm and incubated for 5 minutes at 25° C. 750 ml potassiumacetate buffer (see above) is added while stirring and the temperatureof the biomass is lowered as rapidly as possible to 4° C.

The biomass is centrifuged for 30 minutes at 26000×g and 4° C. Thesupernatant which contains the plasmid DNA is decanted and filteredclear over a 5 μm filter candle.

Chromatography on Q-Sepharose ff:

TE buffer: 10 mmol/l Tris-HCl, 1 mmol/l EDTA pH 8.0±0.2

Equilibration/wash buffer=gradient buffer A: 10 mmol/l Tris-HCl, 1mmol/l EDTA, 0.65 mol/l NaCl pH 8.0±2.

Gradient buffer B: 10 mmol/l Tris-HCl, 1 mmol/l EDTA, 0.85 mol/l NaCl pH8.0±0.2.

The decanted centrifuge supernatant is adjusted to 49-50 mS/cmconductivity by addition of ca. 350 ml TE buffer/l centrifugationsupernatant and cooled to 5°±4° C. The whole chromatography is carriedout at this temperature. The centrifugation supernatant is applied tothe equilibrated column at 5-8 column volumes (CV)/h. Subsequently thecolumn is washed at a flow rate of 5-8 CV/h with ca. 8 CV 10 mmol/lTris-HCl, 1 mmol/l EDTA, 0.65 mol/l NaCl pH 8.0±0.2.

Elution

A gradient is applied to the column (5 CV buffer A, 5 CV buffer B) andthe eluate is fractionated at a flow rate of 5-8 CV/h. The detection iscarried out at 254 nm. The pre-peak (impurities) is separated from themain peak (plasmid DNA) by collecting the main peak from the increasingflank onwards in a separate vessel. The endotoxin content of the eluateis between 1200 and 12000 EU/mg plasmid DNA.

Chromatography on Hydroxylapatite (HA Ceramic)

The chromatography is carried out at 5±4° C.

-   Equilibration buffer: 0.1 mol/l potassium phosphate, 6 mol/l urea pH    7.0±0.2-   wash buffer 1: 0.15 mol/l potassium phosphate, 6 mol/l urea pH    7.0±0.2-   wash buffer 2: 0.02 mol/l potassium phosphate buffer pH 7.0±0.2-   elution buffer: 0.5 mol/l potassium phosphate pH 7.0±0.2

The detection is carried out at 254 nm with a UV detector/plotter unit.A 1% product solution (plasmid DNA) measured with a calibratedphotometer is used as a calibration solution.

The Q-Sepharose® pool is adjusted to a final concentration of 1.1 mmol/lcalcium chloride and applied to the equilibrated column at a flow rateof 5-8 CV/h.

Subsequently the column is consecutively washed at a flow rate of 5-8CV/h with:

-   1. 0.1 mol/l potassium phosphate, 6 mol/l urea pH 7.0±0.2, until    absorbance is no longer detectable on the detector.-   2. 2-4 CV, 0.15 mol/l potassium phosphate, 6 mol/l urea pH 7.0±0.2-   3. 5 CV, 0.02 mol/l potassium phosphate pH 7.0±0.2

Following the wash steps it is eluted with 0.5 mol/l potassium phosphatebuffer pH 7.0±0.1 at a flow rate of 5-6 CV/h.

The peak is collected, heated to 25° C. and 10% of it's volume of 4mol/l KCl solution is added. Subsequently 0.7 parts by volume (relativeto the volume of the eluate) of isopropanol is added, the solutions aremixed and incubated for 5-10 minutes at 25° C. It is centrifuged for 30minutes at ≧20,000×g, the plasmid DNA being in the precipitate.

20 ml 70% ethanol is added to the precipitate and it is againcentrifuged for 10-15 minutes at ≧20,000×g at 4° C.

The precipitate which contains the plasmid DNA is resuspended in TEbuffer (10 mmol/l Tris-HCl, 1 mmol/l EDTA pH 8.0+0.2) and adjusted to aplasmid concentration of 1 mg/ml. The endotoxin content is typicallyless than 0.06 EU/mg DNA and between 0.01 and 0.06 EU/mg DNA.

The endotoxin content is determined by adding a limulus amoebocytelysate solution (LAL solution) to the solution to be examined.Endotoxins result in a gel formation in this mixture.

No gel formation should occur in the negative control preparations andin the positive control preparations as well as in the sample solutionssupplemented with two λ control standard endotoxin a gel formation mustoccur.

The first dilution step of the solution of active substance for whichthese criteria apply and in which no gel formation occurs is used tocalculate the endotoxin content of the solution of active substancesolution according to the following formula:

E=V×λ(EE/ml)

-   -   E: endotoxin content    -   V: dilution factor    -   λ: lysate sensitivity (EE/ml)

EXAMPLE 2 Plasmid Preparation According to the State of the Art

The plasmid preparation is carried out analogously to Birnboim et al.,Nucl. Acids Res. 7 (1979) 1513-1523 and Meth. Enzymol. 100 (1983)243-255. Accordingly the bacterial cells are lysed in NaOH/SDS in thepresence of RNase. It is centrifuged and the supernatant which containsthe plasmid DNA is processed further. The supernatant is loaded onto apre-equilibrated Qiagen® column.

The bacterial mass is resuspended in 4 ml buffer (100 μg/ml RNase A, 50mmol/l Tris-HCl, 10 mmol/l EDTA, pH 8.0). 4 ml lysis buffer (200 mmol/lNaOH, 1% SDS) is added and it is incubated for 5 minutes at roomtemperature. Subsequently 4 ml neutralisation buffer (3 mol/l potassiumacetate, pH 5.5) is added and it is incubated for 15 minutes at 4° C. Itis centrifuged for 30 minutes at 30,000×g at the same temperature andthe supernatant is processed further. A Qiagen® column is equilibratedwith 4 ml equilibration buffer (750 mmol/l NaCl, 50 mmol/l MOPS, 15%ethanol, pH 7.0, 0.15% Triton®X 100) and the supernatant is applied tothe column. It is washed with 1 mol/l NaCl, 50 mmol/l MOPS, 15% ethanol,pH 7.0 and eluted with 5 ml elution buffer (1.25 mol/l NaCl, 50 mmol/lTris-HCl, 15% ethanol, pH 8.5).

The eluate is precipitated with isopropanol (0.7 vol) and centrifugedfor 30 minutes at 15,000 ×g at 4° C. The DNA pellet is washed in 70%ethanol and again centrifuged. Subsequently the pellet is resolubilizedin 10 mmol/l Tris-HCl, 1 mmol/l EDTA, pH 8.0.

The endotoxin content of such a plasmid preparation is typically300-3000 EU/mg. Using an endotoxin removal buffer according to WO95/21177 and Qiagen news 1/96, p. 3-5 the endotoxin content can befurther reduced to ca. 100 EU/mg.

LIST OF REFERENCES

-   Ausubel, F. M., et al. eds. (1991), Current Protocols in Molecular    Biology, Wiley Interscience, New York-   Birnboim, H. C. and Doly, J., Nucleic Acids Research 7 (1979)    1513-1523-   Birnboim, H. C., et al., Meth. Enzymol. 100 (1983) 243-255-   Chandra et al., Analyt. Biochem. 203 (1992) 169-172-   Cotten et al., Gene Therapy 1 (1994) 239-246-   Dion et al., J. Chrom. 535 (1990) 127-147-   European Patent EP-B 0 268 946-   Fiedler, Lexikon der Hilfsstoffe für Pharmazie und Kosmetik und    angrenzende Gebiete (Vol. 9, 3rd edition, 1989, Editio Cantor, GER)-   Garger et al., Biochem. Biophys. Res. Comm. 117 (1983) 835-842-   Johnson & Ilan, Analyt. Biochem. 132 (1983) 20-25-   McClung & Gonzales, Analyt. Biochem. 177 (1989) 378-382-   Protein Purification Methods, Ed. by Elv. Harries and S. Angal,    Oxford University Press 1989, 238-244-   QIAGEN NEWS 1/96, 3-5-   QIAGEN Plasmid Handbook (Qiagen Inc., Chatsworth, USA)-   Raymond et al., Analyt. Biochem. 172 (1988) 125-133-   Sambrook, J. et al. (1989), Molecular Cloning: A Laboratory Manual,    2nd edition, Cold Spring Harbor Laboratory Press, New York, USA-   WO 95/21177

1. DNA preparation comprising a DNA, proteins in an amount of less thanabout 0.1% DNA and endotoxins in an amount of less than about 1 EU/mgDNA.
 2. The DNA preparation of claim 1, comprising endotoxins of lessthan about 0.06 EU/mg DNA.
 3. The DNA preparation of claim 1, comprisingendotoxins of about 0.01 to 0.1 EU/mg DNA.
 4. The DNA preparation ofclaim 1, wherein the DNA preparation is free of ethidium bromide,phenol, cesium chloride, octylphenolpoly(ethylene glycol ether)_(n)detergents and MOPS buffer.
 5. The DNA preparation of claim 1, whereinthe DNA can be replicated in gram-negative bacteria.
 6. The DNApreparation of claim 15, wherein the gram-negative bacteria isEscherichia coli.
 7. The DNA preparation of claim 1, wherein the DNA isplasmid DNA.
 8. The DNA preparation of claim 7, wherein the plasmid DNAis capable of replication.
 9. A pharmaceutical composition suitable forgene therapy comprising a therapeutically effective amount of the DNApreparation of claim 5 and a pharmaceutically acceptable carrier. 10.The composition of claim 9, wherein the DNA preparation containsendotoxins in an amount of less than about 0.06 EU/mg DNA.
 11. Thecomposition of claim 9, wherein the DNA preparation contains endotoxinsin an amount of about 0.01 to 0.1 EU/mg DNA.
 12. The composition ofclaim 9, wherein the DNA is a plasmid DNA.
 13. The composition of claim12, wherein the plasmid DNA is encapsulated in liposomes.
 14. A methodfor gene therapy of cystic fibrosis caused by the absence of a normalfirst gene or the presence of a defective second gene, comprisingadministering an effective amount of the DNA preparation of claim 18 toa patient in need thereof, wherein the plasmid DNA contains said normalfirst gene or a normal second gene corresponding to the defective secondgene.
 15. A process for making the DNA preparation of claim 1,comprising the following steps (a) providing gram-negative bacteriacontaining said DNA; (b) lysing said bacteria to obtain a lysate,wherein the lysate is a DNA-containing fraction and thereafter (c)chromatographing said DNA-containing fraction on hydroxylapatite inorder to obtain said DNA preparation.
 16. The method of claim 15,further comprising replicating said DNA in gram-negative bacteria afterstep (a) and before step (b).
 17. The method of claim 16, furthercomprising transfecting the gram-negative bacteria with a cloning vectorcontaining said DNA before step (a).
 18. The method of claim 16, furthercomprising eluting said hydroxylapatite in step (c) with a solution ofphosphate, citrate, sulfate or divalent metal ions to obtain said DNApreparation.
 19. The method of claim 15, further comprising, after step(b) and before step (c), filtering the lysate to obtain a filtrate andfractionating said filtrate by gel filtration to obtain saidDNA-containing fraction.
 20. The method of claim 16, further comprising,after step (b) and before step (c), filtering the lysate to obtain afiltrate and fractionating said filtrate by gel filtration to obtainsaid DNA-containing fraction.
 21. The method of claim 17, furthercomprising, after step (b) and before step (c), filtering the lysate toobtain a filtrate and fractionating said filtrate by gel filtration toobtain said DNA-containing fraction.
 22. The method of claim 18, furthercomprising, after step (b) and before step (c), filtering the lysate toobtain a filtrate and fractionating said filtrate by gel filtration toobtain said DNA-containing fraction.